Haemostasis, commonly referred to as blood coagulation or blood clotting, is a key biological response to wounding or injury that prevents excessive blood loss. The biochemical cascade that controls haemostasis in mammals is well understood. A crucial step in this pathway is the activation of prothrombin by a prothrombinase complex to produce thrombin, which in turn activates Factor XIIIa, which cross-links fibrin to form a stable clot (Stubbs & Bode, 1994, Curr. Opin. Struct. Biol. 4 823-32).
In mammals, the prothrombin activator complex in vivo typically consists of a serine proteinase factor Xa and a cofactor Va formed on phospholipid membranes in the presence of calcium ions (Suttie & Jackson, 1977, Physiol. Rev. 57 1). The mammalian prothrombinase complex consists of a cofactor, Factor Va, and a serine protease, Factor Xa. Factor Xa alone activates prothrombin very slowly, however, in the presence of accessory proteins including the nonenzymatic cofactor Factor Va, calcium ions (Ca2+) and phospholipid, prothrombin activation is enhanced many fold. In vivo, Factor Xa binds the phospholipid membrane of blood platelets by gamma-carboxyglutamic acid residues and has preferential cleavage for Arg274-Thr275 followed by Arg323-Ile324 bonds in prothrombin to form thrombin.
Given the importance of controlling blood loss during surgery or following injury or trauma, the identification of regulators that either promote blood clotting or inhibit the dissolution of clots (such as by the fibrinolytic plasmin/plasminogen pathway; Royston et al., 1990, Blood Coagul. Fibrinol. 1 53; Orchard et al., 1993, Br. J. Haematol. 85 596) has become an area of intense interest.
In particular, snake venoms have become useful sources of proteins that can either prevent fibrinolysis or promote blood clotting, as a result of blood loss during surgery, trauma in mammals.
For example, inhibitors of fibrinolysis have been isolated from venom of the Australian common brown snake Pseudonaja textilis (International Publication WO 99/58569). With regard to snake venom-derived prothrombin activators, reference is also made to Chinese Patent 1298017 which discloses prothrombin activators isolated from venom of the Taipan snake Oxyuranus scutellatus: prothrombin activating enzyme (designated Os-II) and activated factor Xa. The Chinese group proposed that to promote haemostasis such as in the case of a bleeding wound, Os-II is optimally added one hour before addition of factor Xa to thereby activate prothrombin. They proposed that the simultaneous action of the two can activate prothrombin and raise the yield of thrombin.
Reference is also made to Joseph et al., 1999, Blood 94 621 which discloses a factor Xa-like prothrombin activator (trocarin) isolated from the venom of the Australian rough-scaled snake Tropidechis carinatus. Trocarin forms a prothrombin activator complex that catalyzes formation of thrombin from prothrombin in vitro in the presence of phospholipid, factor Va and calcium ions.
Current haemostatic agents use bovine or human derived blood product components to replace various factors to prevent fibrinolysis or promote blood clotting, as a result of blood loss during surgery, trauma in mammals. The use of bovine or human derived blood product components may potentially expose patients to viral contamination or other adverse events.